The broad, long-term objective of this research proposal is to understand the processes involved in the biogenesis and maturation of the mammalian sperm acrosome, an organelle essential for fertilization. The first specific aim is to determine when two acrosomal proteins are first synthesized and correlate their synthesis with that of the messenger RNA coding for their polypeptides. This laboratory has previously demonstrated that the synthesis one of the acrosomal components, acrogranin, begins during meiosis and continues into earlier spermogenesis. The second specific aim is examine the function of acrogranin by testing the hypothesis that it is similar to proteins found in other secretory granules and may be involved in the packaging of other components into the acrosome. The third specific aim is to examine the effects of epididymis-associated changes of acrosomal components on their function. First, the modification of the oligosaccharide side-chains of the protease zymogen proacrosin will be examined to determine if these changes affect the activated enzyme's activity. In addition, the stability of the acrosomal matrix of sperm from different regions of the epididymis will be examined since changes in the properties of this matrix may affect the ability of sperm to undergo a complete acrosome reaction. The final specific aim is to examine the targeting of proacrosin and acrogranin to secretory granules of cells of the pituitary line AtT-20 that have been transfected with cDNAs coding for proacrosin and acrogranin. These experiments will utilize the methods of cell biology, biochemistry, and molecular biology to look at the biogenesis of the acrosome. The proteins will be localized to specific cells organelles of cells in sections of testes by immunocytochemistry by light and electron microscopy. Functional assays will look at the enzymatic activity of proacrosin/acrosin and the aggregation characteristics of acrogranin. During the course of the project, cDNAs coding for both proacrosin and acrogranin will be isolated and characterized. These probes will allow us to look at the expression of proacrosin and acrogranin mRNA during spermatogenesis and to determine the deduced amino acid sequences for the proteins. Recombinant constructs will be prepared so that these germ cell proteins can be expressed in somatic cells. Creation of stably transfected cell lines expressing these proteins will allow the more facile examination of the biosynthetic transport of these proteins. The results from these experiments will help explain how acrosomal proteins are transported from the cell cytoplasm to the acrosome. Knowledge of this process may provide new avenues for examining cases of male idiopathic infertility and may open new targets for contraceptive development.